Exclusive Temporal Stimulation of IL-10 Expression in LPS-Stimulated Mouse Macrophages by cAMP Inducers and Type I Interferons

Introduction: Expression of the key anti-inflammatory cytokine IL-10 in lipopolysaccharide (LPS)-stimulated macrophages is mediated by a delayed autocrine / paracrine loop of type I interferons (IFN) to ensure timely attenuation of inflammation. Anti-inflammatory macrophages, characterized by enhanced IL-10 expression, can be also generated by a combination of LPS and a second signal, such as an IgG immune complex, apoptotic cell remnants, or a cAMP inducer.

Materials and Methods: We examined the mechanism of IL-10 induction by cAMP in LPS-stimulated mouse macrophages at the promoter level, and explored the crosstalk between type I IFN signaling and cAMP, using the β-adrenergic receptor agonist, isoproterenol, as a cAMP inducer. In addition to experiments preformed with primary BMDM and with RAW264.7 macrophages, we evaluated the physiological effect of cAMP induction on IL-10 expression in a mouse septic shock model.

Results and Discussion: We show that the cAMP pathway directly up-regulates IL-10 transcription and plays an important permissive and synergistic role in early MyD88-dependent LPS-stimulated IL-10 mRNA and protein expression in mouse macrophages and in a mouse septic shock model. In contrast, the cAMP pathway is unable to amplify the late type I IFN-dependent IL-10 induction in LPS-stimulated macrophages and in-vivo. Yet, silencing of the type I IFN receptor enables isoproterenol to synergize with LPS also at the late phase, implying that autocrine type I IFN activity hinders synergistic augmentation of LPS-stimulated IL-10 expression by cAMP at the late phase. Furthermore, IL-10 expression in LPS-stimulated macrophages is exclusively stimulated by either IFNα or isoproterenol. We identified a set of two proximate and inter-dependent cAMP response element (CRE) sites that cooperatively regulate early IL-10 transcription in response to isoproterenol-stimulated CREB and that further synergize with a constitutive Sp1 site. At the late phase, up-regulation of Sp1 activity by LPS-stimulated type I IFN is correlated with loss of function of the CRE sites, suggesting a mechanism for the loss of synergism when LPS-stimulated macrophages switch to type I IFN-dependent IL-10 expression.

Conclusion: This report delineates the molecular mechanism of cAMP-accelerated IL-10 transcription in LPS-stimulated murine macrophages that can limit inflammation at its onset.